PROJECT SUMMARY/ABSTRACT Somatic mutation of DNA (cytosine-5)-methyltransferase 3A (DNMT3AMut) occurs in >20-30% of acute myeloid (AML) patients making it one of the most frequently mutated genes in this deadly human disease. DNMT3AMut correlates with poor clinical outcome of AML. However, the molecular mechanism by which DNMT3AMut contributes to AML development remains far from clear. We reported that DNMT3AMut promotes cell transformation and establishes a bona fide AML phenotype in mice possessing the initiating RAS mutation. Integrated transcriptome and epigenomic profiling of this new murine AML model revealed that DNMT3AMut binds directly to enhancers of target genes inducing focal DNA hypo-methylation at binding sites. These DNMT3AMut-mediated events result in aberrant activation of a gene-expression program controlling stem cell self-renewal (notably a Meis1 node), anti-differentiation and tumor cell pro-survival. Our discovery-based epigenetic inhibitor screen further identified the DOT1L histone methyltransferase to be essential for DNMT3AMut-induced aberrant gene activation. We hypothesize that DNMT3AMut induces focal DNA hypo- leukemia methylation at gene enhancers promoting DOT1L-dependent activation of self-renewal (notably Meis1), anti- differentiation and tumor pro-survival genes, which collectively contribute to AML pathogenesis. Dissecting the molecular events and mechanism underlying DNMT3AMut -mediated AML progression should provide critical insights into new treatment strategies. Towards this goal, we will use cutting-edge CRISPR/Cas9 technologies to define the causal role for enhancer DNA hypomethylation due to DNMT3AMut in inducing aberrant gene activation and promoting AML determine what DNMT3AMut-activated gene pathways are essential for AML development in mice in vitro and in vivo (aim 1); we will through loss-of-function studies in our established murine AML models (aim 2); and third, in a translational aim, we will use human AML cell lines and primary patient-derived xenograft (PDX) models bearing DNMT3AMut to define effects of DNMT3AMut on epigenomic alteration, gene expression deregulation and malignant growth in human AML (aim 3). We expect to define the DNMT3AMut-induced epigenetic/gene changes in AML and expect to identify the pathway by which DNMT3AMut promotes AML progression. Because certain identified pathways such as DOT1L and Bcl2 are potentially druggable with the existing compounds , completion of our proposed research should not only promote a new mechanistic understanding of DNMT3AMut-associated AML but will also yield innovative therapeutics for the treatment of affected cancer patients.